Patients with type II diabetes (diabetes mellitus II, DM II, insulin resistant diabetes) account for 90-95% of all the patients with diabetes, and their amount increases by 6% annually. By 2025, the amount of the patients with type II diabetes is expected to reach 380 million across the world. Asia now is already the region with the most patients with diabetes. The amount of patients with diabetes in developing countries, such as China and India, increases most rapidly across the world. The latest large-scale epidemiological survey about diabetes in China was conducted in 2002 among 100,000 people according to the diagnostic criteria published by WHO in 1999, showing that the prevalence rates in people older than 18 years in urban and rural areas are 4.5% and 1.8%, respectively. In the past two decades, the prevalence rate of diabetes has a nearly 4-fold increase in China.
Currently, the major therapies for type II diabetes include insulin replacement therapy (insulin and insulin analogue) and oral administration of chemical hypoglycemics (insulin secretagogues which can directly stimulate insulin secretion, such as sulfonylureas and glinides; non-insulin secretagogues such as biguanides, thiazolidinediones and alpha glucosidase inhibitors, wherein the biguanides mainly reduce hepatic glucose output, the thiazolidinediones can improve insulin resistance, and the alpha glucosidase inhibitors mainly delay carbohydrate absorption in the intestines). Although the above-mentioned medicaments can decrease blood sugar, they may cause side effects such as hypoglycemia and weight gain, and progressive loss of the function of pancreatic beta-cells (Nature 2001, 414: 821-827).
GLP-1 (Glucagon-Like Peptide-1), as one of the incretins, simulates the “incretin effect” that decreases blood sugar under physiological conditions, and targets two major pathogenesis of diabetes (insufficient insulin secretion and insulin resistance) with unique therapeutic mechanism. At present, GLP-1 has been approved as the second-line medicament in developed countries (Diabetes Care. 2009, 32: 193-203).
GLP-1 promotes insulin secretion and inhibits glucagon secretion in a blood sugar concentration-dependent manner; that is, when blood sugar concentration is higher than normal level, GLP-1 produces an insulin secretion-promoting effect; and when blood sugar concentration is normal, this insulin secretion-promoting effect diminishes. Therefore, the treatment by exogenous GLP-1 will not induce hypoglycemia side effect due to overdose, which is the most prominent feature of GLP-1 analogues over other insulin secretagogues, as well as insulin and insulin analogues (Diabetologia. 1986, 29: 46-52; J Clin Invest. 1993, 91: 301-307; and J Clin Endocrinol Metab. 2001, 86: 3717-3723).
GLP-1 could control postprandial glucagon secretion by binding to the receptors on pancreatic alpha cells; promote proliferation of pancreatic beta cells, inhibit their apoptosis, and increase their sensitivity to glucose by interacting with pancreatic beta cells, thereby increasing glucose-dependent insulin secretion; reduce hepatic glycogen output by acting on liver; delay gastric emptying and reduce food intake by acting on stomach; and increase satiety and reduce appetite by acting on hypothalamus, thereby resulting in weight loss (Diabetes Care. 2003, 26: 2929-2940; Castroenterology. 2007, 132: 2131-2157; Proc Natl Acad Sci. 1982, 79(2): 345-349; Diabetologia. 1996, 39: 1546-1553; Endocrinology. 2003, 144: 5149-5158; Diabetes. 2002, 51: 5434-5442; Diabetologia. 1993, 36; 741-744; and Lancet. 2002, 359: 824-830).
In addition, GLP-1 may improve pathological defects in patients with type II diabetes, protects pancreatic beta cells and cardiovascular system, and has nerve protection effect. Therefore, GLP-1 can reduce occurrence of complications in patients with diabetes, and its advantages and comprehensive effects in decreasing blood sugar, losing weight, and protecting pancreatic cells and cardiovascular system, will certainly improve its position in the future treatment of type 11 diabetes (Diabetes Care. 1998, 21: 1925-1931; Diabetes Spectrum. 2004, 17: 183-190; Lancet. 2006, 368: 1696-1705; and PLoS ONE. 2011, 6(8): e23570).
Natural GLP-1 has no druggability, since it can be easily inactivated in vivo by endogenous DPP-4 (Dipeptidyl peptidase-4) that removes the N-terminal histidine (His) and alanine (Ala) residues of GLP-1, and has a half-life of less than 2 minutes. Therefore, the medicaments under development need to overcome this problem through various ways. At present, there are mainly two classes of GLP-1-targeting medicaments that are marketed or under development: one is small-molecule medicament that can inhibit the degradation effect of DPP-4 in vivo, the other is modified GLP-1 or GLP-1 analogue that has extended half-life without losing the biological function of GLP-1 (J Biol Chem. 1992, 267: 7402-7405; Drug Dev Res. 2001, 53: 260-267; Diabetes. 2007, 56: 1475-1480; Clin Ther. 2008, 30: 858-867; Diabetes Obes Metab. 2008, 10: 82-90; and Curr Med Res Opin. 2008, 24: 275-286).
GLP-1 analogues, GLP-1 mutants, GLP-1 long-acting formulations or DPP-4 inhibitors that have been successfully marketed or under development, are all originally developed to extend the in vivo half-life of active substances. At present, GLP-1 and most of analogues thereof developed at home and abroad possess similar therapeutic effects, and mainly differ in the action time and immunogenicity. Among those medicaments, the first marketed GLP-1 analogue, Exenatide, is developed by Eli Lilly and marketed in the United States in April, 2005, which is derived from the saliva of Gila monster (Heloderma suspectum) and needs to be administered twice a day by subcutaneous injection. The subsequently marketed Liraglutide, a human GLP-1 mutant developed by Novo Nordisk, is marketed in Europe in April, 2009 and in China in October, 2011, which needs to be administered once a day by subcutaneous injection. The human GLP-1 mutant that binds to a human immunoglobulin IgG Fc section, e.g., Dulaglutide under development by Eli Lilly, which takes advantage of the long circulating half-life of IgG and can be administered once a week, is the optimal one among current similar products (Diabetes Obes Metab. 2011, 13: 302-312).
The present invention relates to a fusion protein in which the amino acid sequence of positions 7 to 37 of a human GLP-1 is fused with a human IgG Fc section, which differs from Dulaglutide in that the human IgG Fc section used in this protein is IgG2 Fc section. From a safety perspective, it offers the following advantages:
1) The human GLP-1 polypeptide has low immunogenicity, and thus does not likely generate antibody during long-term use; and
2) The Fc section of certain IgG subtypes (such as IgG1) may bind to the Fc receptors on the surfaces of macrophages and NK cells, having ADCC (Antibody-Dependent Cell Cytotoxicity) and regulation effects. The Fc section of human IgG2 cannot bind to high-affinity Fc receptor, CD64, or to low-affinity Fc receptors, CD32 and CD16, and thus can reduce its ADCC effect.
In view of the above two advantages, the fusion protein of the present invention can not only reduce immunogenicity, but also avoid the effector function of the Fc section that is not associated with the GLP-1 treatment.
Neonatal Fc receptor (FcRn) can extend the half-life of IgG in blood, maintain a high level of IgG concentration in blood circulation, and keep the dynamic balance of antibody level. FcRn is expressed by vascular endothelial cells in normal adults, and can bind to IgG Fc section. Vascular endothelium is an important position where FcRn protects IgG from being degraded and metabolized. FcRn, which depends on endocytosis, not only absorbs IgG from the extracellular acidic environment, but also involves in IgG circulation and homeostatic regulation within cells. Under physiological conditions, when IgG concentration in serum is lower than normal level, more FcRns bind to Fc and decrease IgG degradation, such that IgG concentration can be maintained, when IgG concentration in serum is higher than normal level, the FcRns on the surface of endothelial cells are saturated, and thus cannot bind to more IgG, thereby enhancing IgG degradation and decreasing IgG concentration in serum. By binding FcRn to Fc to protect the Fc-containing protein from being degraded, the high fusion protein concentration in serum can be maintained in a dynamic balance, thereby extending its in vivo half-life.
The present invention, by means of the unique metabolic pathway of immunoglobulin IgG with slow clearance, use a method of fusing a human GLP-1 polypeptide with an Fc section of the human immunoglobulin IgG2 for expression to produce a fusion protein in which GLP-1 has an in vivo half-life close to that of IgG, while maintaining its biological activity.
The fusion protein can be absorbed by subcutaneous injection, and can be administered once every 1 to 2 weeks by subcutaneous injection and maintained an effective in vivo blood drug concentration for a long time due to its prolonged in vivo half-life (the Fc section of IgG2 type having a longer in vivo half-life than those of IgG4 and IgG1 types) (Nature Biotechnology. 2007, 25(12): 1369-1372). Thus, this fusion protein relieves the patients from the pain regarding frequent injections, improves the therapeutic compliance, and reduces the treatment cost.
Although this approach is feasible for GLP-1 therapy, an antibody would be generated when a fusion protein is administered repeatedly over a prolonged period. Furthermore, in view of the fact that the patients with diabetes have to receive treatment during their lifetime after final diagnosis, if the Fc section of the GLP-1-Fc fusion protein retains undesirable effector function, the resultant GLP-1-Fc fusion protein therapy may have a safety concern. The present invention attempts to overcome the problems of potential immunogenicity and effector activity associated with the use of a GLP-1-Fc fusion protein. The fusion protein of the present invention has various amino acid residue substitutions in both the GLP-1 section and the Fc section. The substitutions provide greater potential to increase in vivo stability, reduce immunogenicity and eliminate effector function.